AU648567B2 - Hybrid plasminogen activators - Google Patents

Description

ANNOUNCEMENT OF THE LA ER PUBUCATION PCT OF INTERNATIONAL SEARCH REPORT" INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 91/19793 C12N 15/58, 9/72, A61K 37/54 A3 (43) International Publication Date: 26 December 1991 (26.12.91) (21) International Application Number: PCT/GB91/00945 (74)Agents: VALENTINE, Jill, Barbara et al.; SmithKline Beecham, Corporate Patents, Great Burgh, Yew Tree (22) International Filing Date: 12 June 1991 (12.06.91) Bottom Road, Epsom, Surrey KTI8 3XQ (GB), Priority data: (81) Designated States: AT (European patent), AU, BE (Euro- 9013345.5 14 June 1990 (14.06.90) GB pean patent), CA, CH (European patent), DE (European patent), DK (European patent), ES (European patent), FI, FR (European patent), GB (European patent), (71) Applicant (for all designated States except US): BEECHAM GR (European patent), HU, IT (European patent), JP, GROUP P.L.C. [GB/GB]; SB-Heusc, Great West Road, KR, LU (European patent), NL (European patent), NO, 4f entf rd-Middlesex T ,TW 9D ARle-, //Aozo SE (European patent), US.

Cou^, ,"y/c./ese (G3.

(72) Inventor; and Inventor/Applicant (for US only) ROBINSON, Jeffery, Published Hugh [GB/GB]; SmithKline Beecham Pharmaceuticals, With international search report.

Great Burgh, Yew Tree Bottom Road, Epsomy, Surrey Before the expiration of the time limit for amending the KT18 5XQ claims and to be republished in the event of the receipt of amendments.

(88) Date of publication of the international search report: 7 23 January 1992 (23.01.92) 64856 7 n 6 (54) Title: HYBRID PLASMINOGEN ACTIVATORS .Pasmid pTRH13 BamHI Y 4.1 Kb Fragment H 37 LTR 4" Mlul MMT 3'

DH

SV 40 E Kb Fragment (57) Abstract Fragment A hybrid fibrinolytic enzyme, its preparation, pharmaceutical compositions containing it and its use in the treatment of thromboembolic diseases, in particular acute myocardial infarction.

WO 91/19793 PCT/GB91/00945 -1- HYBRID PLASMINOGEN ACTIVATORS The present invention relates to a hybrid fibrinolytic enzyme, its preparation, pharmaceutical compositions containing it and its use in the treatment of thromboembolic diseases, in particular acute myocardial infarction.

European Patent No 0009879 discloses derivatives of in vivo fibrinolytic enzymes which are useful therapeutic agents for treating venous thrombosis. The derivatives are characterised by the active catalytic site on the enzymes being blocked by a group whic:h is removable by hydrolysis such that the pseudo first order rate constant for hydrolysis is in the range 10 6 to 10 3 sec 1 EP-0155387 discloses a fibrinolytically active hybrid protein which comprises one chain of a 2-chain protease linked to a chain of a different 2-chain protease, or to the same chain of the same protease; at least one of the chains in the hybrid protein being derived from a fibrinolytically active protease, such that the hybrid protein has a catalytic site essential for fibrinolytic activity which is optionally blocked by a removable blocking group.

EP-A-0297882 discloses a hybrid plasminogen activator which comprises the five kringle domains of plasminogen linked to the B-chain of t-PA or u-PA via an amino acid sequence comprising, respectively, the t-PA cleavage site between residues 275 and 276 and the cysteine residue 264 of t-PA or the u-PA cleavage site between residues 158 and 159 and the cysteine residue 148 of u-PA, the catalytic site of the t-PA or u-PA B-chain being optionally blocked by a removable blocking group.

A large number of possible blocking groups were mentioned in WO 91/19793 PCT/GB91/00945 -2- EP-A-0297882 as suitable, particular emphasis being placed on haloanthraniloyl groups, i.e. the 2-aminobenzoyl group substituted in the 3- or 4- position with a halogen atom and optionally further substituted with one or more weakly electron-withdrawing or electron-donating groups.

Particular blocking groups taught in EP-A-0297882 included 2-aminobenzoyl substituted in the 4-position with fluorine, chlorine or bromine.

It has now been found, surprisingly, that blocking a hybrid plasminogen activator (PA) of the type described in EP-A-0297882 with one particular acyl group (the 4-methoxybenzoyl or p-anisoyl group) affords a derivative with particularly advantageous properties in terms of potency.

According to the present invention there is provided 4-methoxybenzoyl plasminogen 1-541/t-PA 262-527 including one and two chain variants, lysj 8 and glul variants, and mixtures thereof.

As used herein the terms t-PA and plasminogen have the meanings and amino acid numbering system given in EP-A-0297882.

In a preferred aspect the acyl hybrid PA of the invention is the glul variant, especially the single chain form thereof.

The acyl hybrid PA preferably comprises at least 60%, more preferably at least 80% of the preferred glul, single chain variant relative to the other chain variants.

In a further aspect the invention provides glul, single chain plasminogen 1-541/t-PA 262-527. This variant is preferably present at a level of at least 60%, more WO 91/19793 PCT/GB91/00945 -3preferably 80% relative to the other chain variants.

The acyl hybrid PA of the invention may be prepared as generally described in EP-A-0297882.

The unacylated hybrid PA is first prepared by expressing DNA encoding the hybrid PA in host cell and recovering the hybrid PA product.

The DNA is prepared conventionally by the condensation of appropriate mono-, di- or oligomeric nucleotide units as described in EP-A-0297882.

The host cell is prepared conventionally by transformation with a replicable expression vector capable, in the host cell, of expressing the coding DNA.

The choice of vector will be determined in part by the host cell, which may be prokaryotic,-such as E. coli, or eukaryotic, such as mouse C127, mouse myeloma, chinese hamster ovary, fungi e.g. filamentous fungi or unicellular 'yeast' or an insect cell such as Drosophila. The host cell may also be in a transgenic animal. Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses, derived from, for example, baculoviruses or vaccinia.

The preferred glul, single chain form of the hybrid PA may be obtained by appropriate choice of vector, host cell, expression conditions and purification conditions. Thus, for example, Chinese hamster ovary (DXB11) cells may be harvested in serum-free medium to prevent proteolysis.

Although this approach usually produces material with a high content of the glu sc form, it may be necessary, especially in harvests made soon after changing the culture conditions from one of serum-containing to serum-free, to add protease WO 91/19793 PCT/GB91/00945 -4inhibitor(s) e.g. aprotinin, to prevent nicking. Similarly, depending on the nature of the purification scheme utilised, protease inhibitors may be required in all or some of the purification buffers.

The hybrid PA is reacted with a suitable acylating agent in order to prepare the 4-methoxybenzoyl derivative.

The invention therefore also provides a process for preparing the acyl hybrid PA of the invention, which process comprises reacting the hybrid PA with a blocking agent JK in which J is a locating group which mediates binding of the agent to the catalytic site of the enzyme and K is a p-methoxybenzoyl group.

Examples of group J include 4-amidinophenoxy and 2-chloro-4-amidinophenoxy.

A preferred acylating agent is 4-amidinophenyl 4'-methoxybenzoate, normally used in the form of the HC1 salt. This compound is disclosed in EP-0009879.

The blocking reactions are preferrably carried out in aqueous media, at a pH in the range 6.75 to 8.5, more preferably 6.75 to 7.25 and at temperatures in the range 0 C to 30 0 C, more preferably 15 0 C to 30 0

C.

The preferred concentration range for the blocking agent is 0.01 to 2.0 millimolar, and the preferred enzyme concentration range is 1 to 500 micromolar.

It is desirable to carry out the blocking reaction in the presence of enzyme solubilising or stabilising additives such as 1-arginine, 1-lysine or 6-aminohexanoic acid.

WO 91/19793 PCT/GB91/00945 The acyl hybrid PA or the glul, single chain plasminogen 1- 541/t-PA 262-527 of this invention is preferably administered as a pharmaceutical composition for the treatment of thrombotic diseases.

Accordingly the present invention also provides a pharmaceutical composition comprising the acyl hybrid PA or glul, single chain plasminogen 1-541/t-PA 262-527 of the invention in combination with a pharmaceutically acceptable carrier.

The compositions according to the invention may be formulated in accordance with routine procedures as pharmaceutical compositions adapted for intravenous administration to human beings.

Typically compositions for intravenous administration are solutions of the sterile hybrid PA in sterile isotonic aqueous buffer. Where necessary the composition may also include a solubilising agent to keep the hybrid PA in solution and a local anaesthetic such as lignocaine to ease pain at the site of injection. Generally, the hybrid PA will be supplied in unit dosage form for example as a dry powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of protein in activity units, as well as, for the acyl hybrid PA, an indication of the time within which the free protein will be liberated. Where the hybrid PA is to be administered by infusion, it will be dispensed with an infusion bottle containing sterile pharmaceutical grade 'Water for Injection'. Where the hybrid PA or is to be administered by injection, it is dispensed with an ampoule of sterile water for injection. The injectable or infusable composition will be made up by mixing the ingredients prior to administration.

WO 91/19793 PCT/GB91/00945 -6- The quantity of material administered will depend upon the amount of fibrinolysis required and the speed with which it is required, the seriousness of the thromboembolic condition and position and size of the clot. The precise dose to be employed and mode of administration must per force in view of the nature of the complaint be decided according to the circumstances by the physician supervising treatment. However, in general, a patient being treated for a mature, fresh or nascent thrombus will generally receive a daily dose of from 0.01 to 10 mg/kg of body weight either by injection in up to five doses or by infusion.

Within the above described dosage range, no adverse toxicological effects are indicated with the hybrid PA of the invention.

Accordingly, in a further aspect of the invention there is provided a method of treating thrombotic diseases, which comprises administering to the sufferer an effective non-toxic amount of an acyl hybrid PA or glul, single chain plasminogen 1-541/t-PA 262-527 of the invention.

In a related aspect there is provided the use of an acyl hybrid PA or glul, single chain plasminogen 1-541/t-PA 262- 527 of the invention for the manufacture of a medicament for the treatment of thrombotic diseases.

The invention also provides an acyl hybrid PA or glul, single chain plasminogen 1-541/t-PA 262-527 of the invention for use as an active therapeutic substance and, in particular, for use in the treatment of thrombotic diseases.

The following Methods and Examples illustrate the invention.

WVO 91,/19793 PCT/GB91/00945 -7- A. General Methods used in Examples DNA Cleavage In general the cleavage of about l~g of plasmid DNA or DNA fragments was effected using about 5 units of a restriction enzyme (or enzymes) in about 20.1 of an appropriate buffer solution.

(ii) Generation of blunt ends If blunt ends were required they were produced by treating the DNA preparation with DNA Polymerase 1, Klenow fragment as described by Maniatis et al, (Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, 1982) Alternatively, sticky ends were removed by digestion using Mung Bean nuclease (Maniatis et al).

(iii) Generation of 'Sticky ends' using linkers Short kinased oligonucleotide linkers encoding single or multiple restriction sites were ligated onto blunt ended fragments, the linker(s) was digested with the appropriate restriction endonuclease producing the required 'sticky end' necessary for further manipulation (Maniatis et al).

(iv) Ligation of DNA Fragments Ligation reactions were carried out as described in Maniatis et al.

Transformation Transformation of plasmid DNA into E. coli HB101 used competent HB101 supplied by Gibco BRL (Paisley, Scotland), WO 91/19793 PCY/GB191 /00945 -8according to the manufacturers instructions.

(vi) Plasmid preparation Large scale preparation of plasmid DNA and plasmid mini-preparations were carried out as described in Maniatis et al (vii) Isolation of DNA fragments from low-melting-point (LMP) agaroseqels DNA fragments were isolated from LMP agarose gels as described by Maniatis et al. Alternatively the excised gel band was purified using GENECLEAN (Stratech Scientific, London) used according to the manufacturer's instructions.

(viii) Oligonucleotide linkers Oligonucleotides were either purchased or were made on Applied Biosystems 381A DNA Synthesizer according to the manufacturer's instructions and were kinased as described in Maniatis et al.

(ix) Chromogenic substrate assays Hybrid was assayed against the chromogenic substrate S2288 (KabiVitrum, Sweden) at a substrate concentration of ImM in 0.1 M triethanolamine.HCl pH 8.0 at 25 0 C. An SU is defined as the amount of activity that gives an O.D. increase at 405nm of 0.001/min in 0.5 ml substrate in a 1 cm pathlength cell.

In another form of the assay, specifically designed for the WO 91/19793 PCT/GB91/00945 -9semi-quantitative assay of chromatography column fractions, il of each fraction was mixed with 100)il ImM S-2288 (as above) in wells of a microtitre plate and the plate incubated at 37 0 C until such time as yellow colour was visible. The solutions were read at 410nm using a Dynatech MR700 Microplate reader.

Fibrinolytic activity assay The fibrinolytic activity of hybrid plasminogen activator solutions was measured on human plasminogen-containing fibrin plates as described (Dodd, and Carr, K., Thrombosis Res. 1989 55 79 85). Dose-responses of hybrid plasminogen activators had slightly different slopes to those of the tissue-type plasminogen activator standard so all activities are approximate. Activities are expressed in IU with reference to the 2nd International standard for t-PA, Lot 86/670. Based on the activity on fibrin plates the hybrid protein is estimated to have a specific activity of approximately 150,000 250,000 lU/mg protein, equivalent to a molar specific activity, of approximately 1.5 2.5 x 13 IU/mole.

(xi) Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) SDS PAGE was carried out to determine the apparent molecular weight(s) of the hybrid plasminogen activator using essentially the method of Laemmli (Nature 1910 227 680-685).

The activators were identified either by staining for protein or by a fibrin zymography technique (Dodd, I. et al Thromb. Haemostasis 1986, 55 94-97). Using these methods it was possible to determine chain nature (sc v tc) and deduce likely N-termini (Glul v Lys 7 8 but note comments under 'N-terminus' (Section WO 91/19793 PCT/GB91/00945 (xii) Rate constant determinations a) The pEaudo first order rate constant was determined by hydrolysing the acyl-enzyme under physiological conditions, i.e. in isotonic aqueous media at pH 7.4 and at 37 0 C. At regular intervals aliquots were withdrawn and incubated with a chromogenic substrate and the rate of conversion of the substrate measured as indicated above.

The hydrolysis was followed until such time as the rate of conversion of substrate reached a maximum. The rate constant k may then be calculated by plotting: Loge (1-At/Amax) against t where Amax is the maximum rate at which an aliquot converts substrate and At is the rate at which an aliquot coiverts substrate at time t.

Preferably such rate.constants are calculated by computerised non-linear regression analysis, fitting the At and time data to the equation:- At A o (Amax-Ao) (1-e-kt) where A o is the activity of the acyl-enzyme preparation before deacylation.

b) Acyl-enzyme (ca. 20 pmol, 101l1) was added to a solution of S-2288 (0.5ml of 1.0mM in 0.05M sodium phosphate, 0.1M NaCl, 0.01% w/v Tween 80 pH (37 0 C) 7.4) in a spectrophotometer cuvette thermostatted at 37 0

C.

Absorbance readings at 405nm were recorded at 1.0 min intervals for 30 min and on-board software (Beckman Inc.) used to calculate the rate of change of absorbance over each WO 91/19793 PC/ B91 /00945 -11successive 1 min interval. As deacylation proceeded in the cuvette, the rate of change of A405nm increased with time.

Rate data obtained when the absorbance exceeded a value of 0.6 were not used because of the effect of substrate depletion. The set of rate determinations were fitted to the following monoexponential function: f(t) A o (Aiax Ao) x (1-e-kt) where A o is the initial activity of the acyl-enzyme and Amax is the maximum activity possible after deacylation and was determined by deacylation of an aliquot of acyl-enzyme in 0.1M Tris. HC1, 20% w/v glycerol, 0.14M NaCI, 0.01% w/v Tween 80 pH 7.4 at 37 0 C for Ih followed by amidolytic assay with S-2288 under the above conditions phosphate buffer pH 7.4, 37 0 A and K, the first order deacylation rate constant, were treated as unknowns in the fitting process and were derived by non-linear regression analysis on a VAX 11/75 compuiter.

(xiii) Determination of plasma deacylation half-life.

The deacylation half-life for some compounds was determined in human plasma rather than in phosphate buffered saline.

Measurement of plasma deacylation rate of acyl enzymes relies on the property of rapid, irreversible inhibition of hybrid plRsminogen activators in plasma following their deacylation. Acyl enzyme (5nM) was incubated in plasma at pH7.4 and 37 0 C and 100i aliquots removed at various time points.

Acyl enzyme not complexed with inhibitors was isolated from the plasma aliquots by euglobulin precipitation. 18 vol acetic acid (0.01% v/v) was added to the aliquot which was 3' then left on ice for 30 minutes before centrifugation at 1000g for 15 minutes at 4 0 C. Precipitates were dissolved in WO 91/19793 PCr/GB9/00945 -12vol PBS-Tween (0.1M NaCI; 0.05M NaH2PO 4 0.01% w/v Tween pH Fibrinolytic activity in the stabilised precipitate was measured on fibrin plates as described in section bv reference to a serially diluted set of standards added to plasma, precipitated immediately (as above) and processed simultaneously. Incubation time on fibrin plates was sufficiently long (17-20h) to allow deacylation of acyl-enzyme and generation of active material.

The level of fibrinolytic activity measured in the aliquots was equated with the amount of acylated material remaining in the plasma at the time of sampling. A semilog plot of percentage initial theoretical fibrinolytic activity versus time was linear. The deacylation half-life is defined as the time at which 50 per cent of the initial theoretical c,ncentration of acyl enzyme had deacylated.

B. Identification of amino acid residues, N-terminus and chain nature used in the examples Sequences All t-PA numbering as in Pennica et al (1983) Nature 301, 214-221; plasminogen amino acid numbering based on Sottrup-Jensen et al (1978) Atlas of Protein Sequence and Structure Vol. 5, Suppl. 3, p91, National Biomedical Research Foundation, Silver Spring. MD., but updated to include the extra amino acid residue identified by Forsgren, M. et al (1987) FEBS Letters. 213 254-260. Plasminogen nucleotide seqi nces as in Forsgren et al (op. cit.).

(ii) N-terminus of hybrid plasminoqen activators GluI indicates the protein is believed to comprise the native (nascent) pl.sminogen N-terminus i.e. amino acid WO 91/19793 P~T/GB91/00945 -13residues 1 onwards.

Lys7 8 indicates the protein is believed to comprise the processed lys 7 8 N-terminus. Alternative processed N-termini e.g. met 69 and va1 79 are known in nature (Miyashita et al 1988, Haemostasis 18 supp. 1, pp 7-13).

References to lys 78 N=terminal forms herein are understood to include lys, val and met forms.

(iii) Chain nature sc. indicates that the protein is in single chain form.

tc. indicates that the protein is in two chain form.

C. Plasmids used in the examples pTRH37 (EP No 0297882) -expression vector for protein H37 (plasminogen 1-541/t-PA 262-527).

pTRE24 (EP 0207589) expressi-on vector for des(cys 5 1 -asp 8 7 t-PA D. p-amidinophenyl p'-anisate HC1 was prepared as described in EP 00098' WO 91/19793 PCT/GB91/00945 -14- Example 1 Expression of plasminogen 1-541/t-PA 262-527 (H37) in Chinese Hamster Ovary cells The plasmid pSV2dhfr was obtained from the American Type Culture Collection (ATCC 37146: Molec. Cell. Biol. 1: 854-864 1981). The plasmid BPV-MT-Xho was obtained from D.

Hamer (National Institutes of Health, Bethesda, Maryland) and contains a version of the mouse metallothionein-1 gene (Hamer and Walling (1982) J. Mol. Appl. Genet. 1 273-268) in which the BglII site just 5' to the translation start point has been converted to an Xhol site by linkering. The dhfr- Chinese hamster ovary cell line (CHO-DXB11) has been described previously (Urlaub, G. and L.A. Chasin, Proc.

Natl. Acad. Sci. 77 4216-4220).

1.1 Construction of an amplifiable expression vector a. Construction of pTRH69 The plasmid pTRH69 was constructed from two fragments A and B isolated from the plasmid PSV 2 dhfr and the plasmid BPV-MT-Xhol respectively.

Fragment A The plasmid PSV 2 dhfr (Fig. 1) was linearised with EcoRI, blunt ended by infilling and linkered with Xhol linkers. The linearised plasmid was then digested with Xhol and BglII releasing fragment A (a 3.4kb fragment encoding the dihydrofolate reductase cDNA and SV 40 early promoter sequences).

Fragment B The plas:.;id BPV MT-Xhol was linearised with SSstl, blunt-ended by nuclease digestion and linkered using BglII linkers. The linear plasmid was then digested with HindIII, blunt ended by infilling and linkered with Xhul linkers. The plasmid was then digested with BglII and Xhol, re'easing fragment B (0.3kb encoding the 3 WO 91/19793 P(7/GB91/00945 metallothionein polyadenylation sequences) (Fig. 2).

Fragments A and B were isolated by LMP agarose electrophoresis and ligated together. The ligated DNA was transformed into E coli HB101 and the plasmid PTRH69 was obtained (Fig. 3).

b. Construction of The plasmid pTRH69 was linearised with Xhol and phosphatased. The LMP agarose-purified fragment was ligated with a 3.3Kb Xhol fragment derived from the plasmid pTRE24, which encodes a modified t-PA protein. E. coli HB101 was then transformed with the ligated DNA to obtain (Fig. 4).

c. Construction of pTRH13 The plasmid pTRH70 was digested with Mlul and BamHl; a fragment (comprising the vector functions) was isolated by LMP agarose gel purification and ligated with a 4.1Kb Mlul/BamHl fragment isolated from pTRH37 (encoding hybrid protein H37). E. coli HB101 was transformed with the ligated DNA and the plasmid pTRH13 was obtained (Fig.

1.2 Expression of hybrid protein a. Cell preparation CHO cells were trypsinised and plated out at 5 x 105 per dish and left in growth medium (Hams F12 nutrient media (041-01765) with 1% stock glutamine (043-05030), 1% stock penicillin/streptomycin (043-05070) and 10% bovine foetal calf serum (011-6290); Gibco, Paisley, Scotland) at 37 0 C in a humidified incubator in an atmosphere of 5% C0 2 /95% air.

Tfter hrs the r Pll were nri~& for nMi tr=ncfe'tinn WO 91/19793 PC/GB91/00945 -16b. Transfection procedure The transfection procedure, carried out in growth medium used calcium coprecipitation and glycerol shock as described in: DNA Cloning, Ed D.M. Glover (Chap. 15. C. Gorman).

Following transfection with pTRH13 the cells were maintained in growth medium for 46 hrs under growth conditions (as above) prior to the selection procedure.

c. Selection and Co-amplification The selection and co-amplification procedure was carried out essentially as described by R. J. Kaufman (1985) Molecular and Cellular Biology 5, 1750-1759. 46hrs post transfection the cells were medium changed into selective medium MEM ALPHA (041-02571), 1% stock glutamine (043-05030), 1% stock penicillin/streptomycin (043-05070) and 10% dialysed bovine foetal calf serum (220-6300AJ) (Gibco, Paisley, Scotland).

The cells were maintained in selective medium for 8-10 days until colonies of dhfr+ cells appeared. When colonies were established the cells were medium changed into selective medium containing methotrexate, (A6770; Sigma, England).

The methotrexate concentration was initially 0.02NM and was increased stepwise to 5p1M. Early batches of the hybrid protein H37 were produced from a mass culture (CHOZ3) amplified to 31.M methotrexate. Later batches were produced either from two cell lines (CHOZ5-C4 and CHOZ5-C13) which were isolated by ring cloning from a mass culture amplified to 5pM methotrexate (CHOZ5), or CHOZ5-C4E12, which is a subclone (dilution plated) of CHOZ5-C4.

d. Detection of hybrid protein H37 During the amplification procedure aliquots of growth medium from growing cells were assayed for plasminogen activator WO 91/19793 PCT/GB91/00945 -17production by fibrin plate and zymography as described in Methods. Zymography revealed a fibrinolytically active protein with apparent Mr approximately 100,000, which is consistent with the expected molecular weight of hybrid protein H37.

e. Purification of hybrid protein H37 CHO cells transfected with pTRH13 (Example l.lc) and selected for survival in 31M methotrexate (Example 1.2c) were harvested (100mls per 175cm 2 flask) for 3 or 4 days in Hams F12 nutrient mixture: Dulbecco's modified Eagles medium (50:50)and 1% pencillin/streptomycin (GIBCO, Paisley, Scotland) (note, serum free).

5.81 of conditioned medium from a typical 3d harvest was collected and was purified on zinc chelate Sepharose (vt 240ml) and lysine Sepharose (vt60ml) using essentially the same method as that described in Dodd, I. et al (FEBS Lett., .986 209, 13) except.that the lysine Sepharose column was eluted with two arginine-containing buffers rather than just one. The first was 0.02M Tris/0.5M NaCl/0.25M L-arginine/0.01% Tween 80 pH 7.4 and the second was 0.02M NaCl/0.5M L-arginine/0.01% Tween 80 pH 7.4. They were applied sequentially in the order given here. The eluate during these elutions was fractionated. The hybrid-containing fractions were identified with a rapid microtitre plate-based assay using S2288. (In this and many other experiments the hybrid eluted in the 0.5M arginine wash. However, in other experiments the hybrid elutes in the 0.25M arginine wash; the reason is not known but appears to be related to the loading level i.e. mg hybrid bound per ml of gel of the lysine Sepharose.) They were pooled and ultrafiltered at 4 0 C using a membrane with a molecular weight cut-off of 10,000 (Amicon, 'YM The ultrafiltered retentate (10.7ml) contained approximately WO 91/19793 PCT/GB91/00945 -18- 220,000 IU/ml and 23,000 SU/ml. These figures generate an IU:SU ratio of 9.6 (but see comments in Methods:assay); other purifications typically yielded good quality sc H37 with lower ratios. Analysis by SDS PAGE under reducing conditions followed by staining for protein using Page Blue 83 (BDH) indicated the retentate contained at least 80 per cent glul sc hybrid protein. The retentate was stored at 0

C.

Example 2 Preparation of two chain plasminoqen 1-541/t-PA 262-527 (tcH37) Single chain H37 (10mg ml 1 in 0.02M Tris/0.5M L-arginine/0.01% Tween 80 pH 7.4; 0.5ml) isolated from CHOZ5-C4 and -C13 cell lines was mixed with human lys plasminogen (0.5mg ml 1 barbitone/saline buffer; 31l) and incubated for 2h at 37 0 C and then stored at -40 0

C.

Analysis by SDS PAGE. followed by protein staining and fibrin zymography showed that the majority of Lhe incubate was'in the glul two-chain form.

Example 3 Preparation of two chain plasminogen 78-541/t-PA 262-527 (tc Ivs 7 8 H37) The preparation of tc lys 78 H37 was the same as that described above (Example 2) except that the stock lys plasminogen was at 5mg ml 1 so that the final plasminogen concentration was 10-fold higher. The subsequent analyses confirmed that the material was mainly in the lys 78 two chain form.

WO 91/19793 PCT/GB91/00945 -19- Example 4 Synthesis of p-anisoyl single chain plasminogen 1-541/t-PA 262-527 Purified sc H37 isolated from mainly the CHOZ3 mass culture nmoles in 1.95ml 0.02M Tris/0.2M NaCl/0.2M L-arginine/0.01% Tween 80pH 7.4) was mixed at 25 0 C with p-amidinophenyl-p'-anisate. HC1 (100mM in DMSO, 401l) and incubated at 25 0 C for lh. Assay of the incubate using the chromogenic substrate S-2288 revealed 99% inhibition of the activity present at t 0. To remove excess acylating agent, the 2.0ml was buffer-exchanged into 0.02M Tris/0.2M NaCl/0.2M L-arginine/0.02% Tween 80 pH 7.4 using Sephadex (PDIO) and the product stored at -40 0

C.

The solution was thawed and an aliquot diluted in 0.05M sodium phosphate/0.1M NaCl/0.01% Tween 80 pH 7.4 at 370C.

Deacylation was measured using S-2288. Under the above conditions the deacylation half-life in this buffer was 33±2 min, equivalent to a deacylation rate constant of 3.5 x 10 4

-I

sec-.

Example Synthesis of p-anisoyl two chain plasminogen 1-541/t-PA 262-527 Two chain H37 was prepared as described in Example 2. 23 nmoles in 1.5ml 0.02M Tris/ 0.5M NaCI/ 0.5 M L-arginine/0.01% Tween 80 pH 7.4 was mixed at 25 0 C with p-amidinophenyl p'-anisate. HCI (100mM in DMSO, 301) and incubated at 25 0 C for 30 min. Assay of the incubate using S-2288 revealed 99% inhibition of the activity present at t 0. The mixture was buffer-exchanged into 0.02M Tris/0.2M NaCl/0.2M L-arginine/0.01% Tween 80 pH 7.4 using a Sephadex WO 91/19793 PC/GB91/00945 PD10 column. The buffer-exchanged product was stored at -40 0 C. The deacylation half-life in human plasma at 37 0

C

was 35 min.

Example 6 Synthesis of p-anisoyl two chain plasminoaen 78-541/t-PA 262-527 Two chain lys 7 8 H37 was prepared as described in Example 3.

21 nmcles of tc lys 78 H37 in 1.5ml 0.02M Tris/ NaC1/ 0.5M L-arginine/0.01% Tween 80 pH 7.4 was mixed at 0 C with p-amidinophenyl p'anisate. HC1 (100mM in D..SO; and incubated at 25 0 C for 30 min. Assay of the incubate using S-2288 revealed 99% inhibition of the activity present at t 0. The mixture was buffer-exchanged into 0.02M Tris/0.2M NaCl/0.2M L-arginine/0.01% Tween 80 pH 7.4 using a Sephadex G25 PD10 column. The buffer-exchanged product was stored at -40 0 C. The deacylation half-life of the product in human.plasma at 370C was 34 min.

Sephadex G25 is a Trademark PCr/GB91/'O945 WO 91/19793 -21- Legends to Figures Fig 1 Plasmid PSV 2 dhfr P Simian virus polyadenylation sequence)

SV

4 0 3' I Simian virus 40 intron sequence

DH

SV

40

E

EcoRI BglII dihydrofolate reductase cDNA Simian virus 4 0 early promoter Restriction enzyme site: EcoRI was linkered with Xhol linkers after EcoRI digestion SRestriction enzyme site: TglII Fiq 2 Plasmid BPV-MT-Xhol

BPV

MT-1 Sstl Bovine papillomavirus sequences SMouse metallothionein gene sequences including 3' polyadenylation sequence (0.3Kb fragment B) Restriction enzyme site: Sstl was linkered with BglII linkers after SstI digestion Restriction enzyme site: HindIII was linkered with Xhol linkers after HindIII digestion HindIII WO 91/19793 PTG9/04 PCr/GB91/00945 -22- Plasmid pTRH69 MMT 3'

DH

SV

4

OE

-Mouse metallothionein polyadenylation sequences: Fragment B excised from plasmid BPV-MT-Xho 1 -dihydrofolate reductase cDNA Fragmrent A -Simian virus 40 early promoter) excised firom.

plasmid PSV 2 dbr Xhol Restriction enzyme site Xhol BglII =Restriction enzyme site BglII Fig 4 Plasmid

LTP,

Sv 4 0 3'

MUT

-Rous sarcoma virus long terminal) 3.3Kb repeat (XhoI) -Simian virus 40 intron and fragment polyadenylation sequences from PTRE24 -t-PA Mutein cDNA -Mouse metallothionein poiyadenylaticn secquences -dihydrofolate reductase cDNA Simian virus early promoter MluI, XhoI: indicate restriction site:s MMT3'

DH

SV

4

OE

Bara HI, WO 91/19793 PTG9/04 PCr/GB91/00945 -23- Fig Plasmid pTRH13 LTR =Rous sarcoma virus long terminal) 4.1Kb (B,-rn repeat HI-MiuI)

SV

40 31 Simian virus 40 intron and fragment polyadenylation sequences from pTRH37 Ht37 =H37 hybrid protein cDNA MMT31 Mouse metallothionein polyadenylation sequences DH Dihydrofolate reductase cDNA

SV

40 E =Simian virus 40 early promoter BamHI, MiuI: indicate restriction sites

Claims (9)

1. 4-Methoxybenzoyl plasminogen 1-541/t-PA 262-527 including one and two chain variants, lys 7 e and glu, variants, and mixtures thereof.

2. 4-Methoxybenzoyl plasminogen 1-541/t-PA 262-527 glu, single chain variant.

3. A compound according to any preceding claim, substantially as hereinbefore described with reference to the foregoing Examples.

4. A pharmaceutical composition which comprises a compound according to any one of claims 1 to 3 in combination with a pharmaceutically acceptable carrier.

A compound according to any one of claims 1 to 3 for use as an active therapeutic substance.

6. A compound according to any one of claims 1 to 3 for use in the treatment of thrombotic diseases.

7. Use of a compound according to any one -f claims 1 to 3 for the manufacture of a medicament for the Creatment of thrombotic diseases.

8. A method of treating thrombotic diseases which comprises a-dminisxeriiiy to the sufferer an effective non-toxic amount of a compound according to any one of claims 1 to 3.

9. A process for preparing a compound according to claim 1 or 2, which comprises reacting plasminogen 1-541/t-PA 262-527 including one and two chain variants, lysy 7 and gl,. variants, and mixtures thereof with a blocking agent JK in which J is a locating group which mediates binding of the agent to the catalytic site of the enzyme and K is a p-methoxybenzoyl 940215,p,\opr\]mw,806819cL46,24 25 group. DATED this FIFTEENTH day of FEBRUARY 1994 Beecham Group p.l.c. By DAVIES COLLISON CAVE Patent Attorneys for the Applicant(s) 940215 SP:\c'ner~bxmw,80681t91cL46,25